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|Title:||Investigating the mechanism and energy coupling of DNA gyrase|
|Authors:||Smith, Clare Victoria.|
|Presented at:||University of Leicester|
|Abstract:||DNA gyrase is the bacterial type II topoisom erase w hich couples the free energy o f ATP hydrolysis to the introduction of negative supercoils into DNA. The active enzym is composed of two GyrA and two GyrB subunits forming an A2B2 complex. The specific supercoiling activity of GyrB was found to be consistently lower than the specific supercoiling activity of GyrA and this is believed to be due to mis-folding of the subunit. Expression as a thioredoxin-fusion protein did not improve the specific supercoiling activity of GyrB. The C-terminal 47 kDa domain of GyrB (GyrB47) was over-expressed as a soluble protein when fused to thioredoxin. This domain interacts with GyrA and DNA. In complex with GyrA, GyrB47 supports quinolone- and Ca2+-induced DNA cleavage and has ATP-independent relaxation activity which is comparable with that of full-length GyrB. GyrB47 also supports low level ATP-independent decatenation.;Protein cross linking was used to investigate nucleotide-, DNA- and drug-induced conformational changes during the reaction cycle of the enzyme. Upon addition of the non-hydrolysable ATP analogue, ADPNP, there was an increase in crosslinking between the GyrB subunits. In the presence of DNA, crosslinks between the GyrA subunits were identified. Using limited proteolysis and immunodetection, these crosslinks were shown to be between the N-terminal 64 kDa domains of GyrA.;Examination of the X-ray crystal structure of the 43 kDa domain of GyrB (GyrB43) shows that the side chains of Gln335 and Lys337 interact with the gamma-phosphate of the ATP. Both these residues are highly conserved among type II topoisomerases. The proposed role of these residues is in nucleotide binding, transition-state stabilisation and triggering conformational changes following ATP hydrolysis. Site-directed mutagenesis was used to convert Gln335 to Asn and Ala and Lys337 to Gln and Ala. No clear role for Gln335 has been established in nucleotide binding, ATP hydrolysis or triggering conformational changes. However mutations at Lys337 lead to a modest decrease in nucleotide binding and a large reduction in ATP hydrolysis (~103-fold decrease in k cat). Therefore Lys337 is a critical residue for ATP turnover and the results are consistent with its involvement in transition-state stabilisation.|
|Rights:||Copyright © the author. All rights reserved.|
|Appears in Collections:||Theses, Dept. of Biochemistry|
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